Determination of neomycin sulfate and impurities using high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Neomycin B is one of a class of aminoglycoside antibiotics that lack a good chromophore, and is therefore difficult to determine using reversed-phase HPLC with absorbance detection. This is especially true for determining the quantity of each impurity. We show that neomycin sulfate and its major impurities, including neamine (neomycin A), can be separated on a strong anion-exchange column using a weak potassium hydroxide eluent (2.40 mM) at a column temperature of 30 degrees C, and directly detected by integrated pulsed amperometric detection (IPAD). The resolution (United States Pharmacopeia (USP) definition) between neomycin B and the closest major impurity ranged from 6.56 and 7.45 over 10 days of consecutive analysis (7.24+/-0.10, n=836 injections). Due to the difficulty of producing weak hydroxide eluents of the required purity (i.e. carbonate-free), this method depends on automatic eluent generation to ensure method ruggedness. This method exhibited good long-term (10 days, 822 injections) retention time stability with a R.S.D. of 0.6%. Peak area R.S.D. (10 microM) was 1.3%. Method robustness was evaluated by intentionally varying the flow rate, eluent concentration, column temperature, and column. The spike recoveries of neomycin B from extractions of three different topical ointments and cream formulations ranged from 95 to 100%. The measured concentration of neomycin B in these formulations ranged from 119 to 154% of the label concentration. The R.S.D. for the measured concentration of one of the formulations tested over three separate days, n=11 extracts, was 3.2%. Based on the results of these evaluations, we believe this method can be used for neomycin sulfate identity, assay, and purity.     

A high-performance liquid chromatographic assay for clindamycin phosphate and its principal degradation product in bulk drug and formulations

A high-performance liquid chromatography method has been developed for the analysis of clindamycin phosphate and clindamycin, the principal degradation product. The method is quantitative, precise and is able to separate a variety of closely related molecules. The method has been applied to bulk drug, topical and sterile solutions, and experimental cream, lotion and gel formulations. The method gives results that are in good agreement with the official gas chromatographic method but is much less time-consuming.     

Stress degradation study on sulfadimethoxine and development of a validated stability-indicating HPLC assay

Sulfadimethoxine was subjected to different International Conference on Harmonisation prescribed stress conditions (hydrolysis, oxidation and photolysis). A stability-indicating high-performance liquid chromatography method was developed for analysis of the drug in the presence of its major degradation products. It involved a Knauer Eurospher C18 thermostated column at 25 °C, and 9.57 mM phosphate buffer (pH adjusted to 2.0 with orthophosphoric acid)-acetonitrile (70:30 v/v) as mobile phase. The mobile phase flow rate and sample volume injected were 1.2 mL/min and 20 μL respectively. The selected wavelength for the determination was 248 nm. The method was validated for its linearity, precision, accuracy, specificity and selectivity, and then applied to the assay of sulfadimethoxine in pharmaceutical formulations. The results of the study show that sulfadimethoxine is highly sensitive to basic hydrolysis and oxidation. The mechanisms and schemas of hydrolytic, oxidative and photolytic degradation are also studied. The method developed, which separates all of the most degradation products, is simple, accurate, precise and specific. Thus, it can be applied to study the stability of veterinary preparations containing sulfadimethoxine.     

Identification of tobramycin impurities for quality control process monitoring using high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Commercial-scale fermentation for tobramycin manufacture is carried out with Streptomyces tenebrarius. Impurity profiling during various phases of pharmaceutical production is important for evaluating the effectiveness of a processing step and meeting regulatory requirements. High-performance anion-exchange (HPAE) chromatography with integrated pulsed amperometric detection (HPAE-IPAD) is a highly sensitive method used to assay tobramycin and to assess purity, but no prior publications demonstrated the capability of this technique to monitor purity at various stages of production at either the typical concentrations or in the typical matrices of a manufacturing process. In addition, the identities of the impurity peaks observed in commercial sources of tobramycin when assayed by using HPAE-IPAD are mainly unknown. Regulatory agencies generally require these impurities to be characterized when found above certain limits, and when present at higher levels require toxicological studies. In this paper, we analyze tobramycin samples using HPAE-IPAD at different stages of production and show the impurity profile and concentration changes through the manufacturing process. We successfully identified nearly all the impurity peaks found in commercially available tobramycin, based on known degradation pathways deduced from extreme pH forced degradation studies, which we experimentally reproduced, and based on previously known related substances found in S. tenebrarius fermentation broth. In crude and final tobramycin products, we identified the peaks for neamine, kanamycin B, nebramine, kanosamine, 2-deoxystreptamine. We tentatively identified deoxystreptamine-kanosaminide in crude and final products, and kanamycin A, carbamoyl-kanamycin B and carbamoyl-tobramycin in down stream process intermediates of a S. tenebrarius fermentation culture. Results presented in this paper support the effective use of the HPAE-IPAD method for in-process impurity profiling of tobramycin, and as a stability-indicating technique after product purification.     

曲安奈德新霉素贴膏的质量评价

目的评价国内不同企业生产的曲安奈德新霉素贴膏的质量。方法按照现行质量标准检验结合探索性研究,包括醋酸曲安奈德及硫酸新霉素鉴别方法的改进、醋酸曲安奈德和硫酸新霉素的含量测定方法的建立、含量均匀度的测定、残留溶剂的考察等,综合评价产品质量及现行质量标准对产品质量的可控性。结果抽取的267批样品,按现行国家标准检验合格率为98.9%。探索性研究结果表明,改进的鉴别方法专属性及可操作性均较高,能更好的鉴别两个主成分;不同厂家的曲安奈德新霉素贴膏两个主成分含量水平存在较大差异,抽取6个厂家的样品中有5个厂家硫酸新霉素含量不合格且均一性较差;1个厂家溶剂残留量超出规定范围。结论目前曲安奈德新霉素贴膏总体质量一般;现行标准不能很好控制产品质量,需进一步提高,建议现行标准改进两个主成分醋酸曲安奈德及硫酸新霉素的鉴别方法,增加含量测定项目;建议企业进一步优化生产工艺,控制样品含量均匀性及溶剂残留量,以提高产品质量。     

Feasibility of Measuring the Bioavailability of Topical Betamethasone Dipropionate in Commercial Formulations Using Drug Content in Skin and a Skin Blanching Bioassay

An in vivo technique has been developed which simultaneously compares a skin blanching bioassay with drug content in human stratum corneum following topical application of four 0.05% beta-methasone dipropionate formulations. Bioavailability of drug from commercial cream and ointment formulations was assessed by quantification of drug content in tape-stripped stratum corneum and skin blanching in the treated skin site under occluded conditions. Tape-stripping removed stratum corneum to a varying degree between individuals but was consistent (35%) within an individual with all formulations, day to day. A correlation (r = 0.9935) between the amount of drug in the treated stratum corneum normalized for surface area and the corresponding skin blanching score was observed with four 0.05% betamethasone dipropionate formulations. Increasing the amount of drug in the tape-stripped stratum corneum correlated with an increased skin blanching score. Ointment formulations delivered more drug to the skin and produced greater blanching scores than the cream formulations. Topical corticosteroid content in the treated skin site can therefore be quantified and correlates well with the resulting pharmacodynamic activity.     

曲咪新乳膏质量分析

目的 通过对20家生产企业355批曲咪新乳膏全国抽验结果分析,评价曲咪新乳膏产品的质量状况和现行质量标准 的可行性。方法 通过法定标准检验,结合检验结果、实验过程及现象,在药品安全性、稳定性及有效性等方面开展探索性研 究,综合评价其质量状况。结果 法定标准检验355批次样品,352批次符合规定,合格率为99.2%。探索性研究显示,由于处 方或工艺控制的不合理,导致某些企业样品出现硫酸新霉素降解,曲安奈德杂质含量增加等问题。探索性研究还发现部分企业 样品中检出处方中未标注的辅料。结论     

曲咪新乳膏质量分析

目的 通过对20家生产企业355批曲咪新乳膏全国抽验结果分析,评价曲咪新乳膏产品的质量状况和现行质量标准 的可行性。方法 通过法定标准检验,结合检验结果、实验过程及现象,在药品安全性、稳定性及有效性等方面开展探索性研 究,综合评价其质量状况。结果 法定标准检验355批次样品,352批次符合规定,合格率为99.2%。探索性研究显示,由于处 方或工艺控制的不合理,导致某些企业样品出现硫酸新霉素降解,曲安奈德杂质含量增加等问题。探索性研究还发现部分企业 样品中检出处方中未标注的辅料。结论 目前曲咪新乳膏的总体质量状况较好,但现行质量标准有提高空间。     

Quantitative determination of hexamidine isethionate in pharmaceutical preparations by high-performance liquid chromatography

Hexamidine isethionate in various pharmaceutical formulations was analyzed quantitatively by high-performance liquid chromatography. The method is rapid, accurate, and precise. Excellent results were obtained from four commercial bases.     

High-pressure liquid chromatographic determination of saccharin in artificial sweeteners and pharmaceuticals.

A high-pressure liquid chromatographic procedure is presented for determining saccharin in various formulations. The method is fast, precise, and accurate and is specific for saccharin in the presence of its most likely impurities and degradation products. Reversed-phase chromatography on a micro-C18 column is utilized with an internal standard, and detection is by UV absorption at 280 nm.     

曲安奈德新霉素贴膏中硫酸新霉素含量测定方法的建立

目的 建立HPLC法测定曲安奈德新霉素贴膏中新霉素含量测定的方法。方法 首先,通过正己烷溶解膏剂,水萃取的方法提取贴膏剂中的硫酸新霉素,然后建立HPLC法测定其含量,色谱条件为：Apolllo C18色谱柱(250mm×4.6mm, 5μm),流动相：0.1mol/L三氟醋酸溶液-乙腈(80:20,V/V),流速：0.5mL/min,进样量：20μL,柱温为30℃;蒸发光散射检测器：漂移管温度：80℃,载气流速：2.0L/min,增益为1。结果 硫酸新霉素在1.0~5.0μg范围内,峰面积与样品浓度呈良好的log-log线性关系,线性方程为：Y=1.87X+7.12(r=0.999),定量限为：0.260μg,平均回收率为88.99%,重复性RSD为1.9%。结论 所建立的方法简便、准确且快速,可用于曲安奈德新霉素贴膏中硫酸新霉素含量测定。     

An HPLC method for quantitation of perillyl alcohol in a topical pharmaceutical cream formulation

A reverse phase high performance liquid chromatographic method for quantitation of perillyl alcohol in a topical cream pharmaceutical formulation was developed. Previously reported methods for analyzing drugs in lipid formulations are relatively complex and time consuming, with extraction, purification and derivatization involved. Through a simple dilution of the cream formulation in isopropyl alcohol, the present assay method enables the direct injection of the samples, on an Alltima C18 5 mu, 150 mm x 2.1 mm, narrow bore column (Alltech Associates, Deerfield, IL). The method includes an isocratic run with acetonitrile-water (40:60, v/v) at 0.35 mL/min for 12 min, followed by a gradient wash with isopropyl alcohol for 20 min, to ensure that all formulation excipients are eluted. Ultraviolet detection was performed at 210 nm with a retention time for perillyl alcohol of 7 min. The high sensitivity assay utilizes a small (5 microL) injection volume for the accurate and precise analysis of perillyl alcohol from a complex cream formulation.     

Stability-indicating LC assay of and impurity identification in homoharringtonine samples

Homoharringtonine (HHT) is a potent myelosuppressive agent and has antitumor activity. Recent studies suggest that it inhibits tumor growth by inducing apoptosis. HHT is an ester of the alkaloid cephalotaxine. It is isolated from genus Cephalotaxus. At least ten HHT analogs have been identified from cephalotaxus extracts. High performance liquid chromatography (HPLC) separations of the cephalotaxine alkaloids in plant extracts have been reported, they have not been validated as specific and stability-indicating for HHT. Due to the complexity of the alkaloid extracts, it is conceivable that additional analogs may still be unresolved from HHT. This paper presents an improved and validated HPLC assay for HHT. The assay is stability-indicating, precise (R.S.D. < 1%), linear (r2 = 0.9999), and accurate (error < 1%). The assay reveals three congeners present as impurities in HHT samples. Two are new and have not been previously reported. Identities of the impurities and forced decomposition products, elucidated with their HPLC retention and spectral data, are also presented.     

Evaluation of enzymic assay for paracetamol: clinical and forensic experiences

The performance of the enzymic paracetamol assay (Cambridge Life Science, UK) was evaluated for use in clinical and forensic analyses and compared with gas—liquid chromatography and EMIT (Syva) systems. It was found to be precise, accurate and rapid for the analysis of paracetamol in serum, with a possible application for the quantitative screening of post mortem blood.     

LC-UV method development and validation for the investigational anticancer agent imexon and identification of its degradation products

Imexon (4-imino-1,3-diazabicyclo[3,1,0]-hexan-2-one) is a member of the class of 2-cyanoaziridine derivatives, which have been of interest as immunomodulators and anticancer agents since the late 1970s. The pharmaceutical development of imexon necessitated the availability of an assay for the quantification and purity determination of imexon active pharmaceutical ingredient (API) and the drug in its pharmaceutical dosage form. A liquid chromatographic (LC) method with ultraviolet (UV) detection was developed, using a reverse phase column with phosphate buffer (pH 6; 50 mM) as mobile phase and UV detection at 230 nm. Although retention capacity for imexon was small (capacity factor of 0.5), the method was found to be linear over the concentration range of interest of 1.0-25 microg/mL, precise, accurate, and stability-indicating. Moreover, the use of LC-mass spectrometry (MS) and on-line photodiode array (PDA) detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API. The degradation products, including chloro- and hydroxy-derivatised products were shown to arise from nucleophilic reactions with the activated aziridine moiety of imexon. The developed LC-UV method was found suitable for the pharmaceutical quality control of imexon API and the drug in its pharmaceutical dosage form.     

High-performance liquid chromatography assay for the determination of the HIV-protease inhibitor tipranavir in human plasma in combination with nine other antiretroviral medications

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay has been developed and validated for the simultaneous quantitative determination of tipranavir with nine other antiretroviral drugs in plasma. A liquid-liquid extraction of the drugs in tert-butylmethylether (TBME) from 200 microL of plasma is followed by a reversed phase gradient HPLC assay with UV detection at 210 nm. The standard curve for the drug was linear in the range of 80-80,000 ng/mL for tipranavir; 10-10,000 ng/mL for nevirapine, indinavir, efavirenz, and saquinavir; and 25-10,000 ng/mL for amprenavir, atazanavir, ritonavir, lopinavir, and nelfinavir. The regression coefficient (r(2)) was greater than 0.998 for all analytes. This method has been fully validated and shown to be specific, accurate and precise. Due to an excellent extraction procedure giving good recovery and a clean baseline, this method is simple, rapid, accurate and provides excellent resolution and peak shape for all analytes. Thus this method is very suitable for therapeutic drug monitoring.     

A validated HPLC assay for salmon calcitonin analysis. Comparison of HPLC biological assay

A high-performance liquid chromatographic (HPLC) method is described for the assay of salmon calcitonin. The method uses a 5-μm octadecasilyl silica column (100 × 4.6 mm) at 50°C and an initial mobile phase (flow rate 1 ml min⁻¹) comprising 35% of B (1 M tetramethylammonium hydroxide—water—acetonitrile, 8:392:600) and 65% of A (1 M tetramethylammonium hydroxide—water—acetonitrile, 20:880:100) with linear gradient elution over 21 min to a final mobile phase of 57% B; solutions A and B are adjusted to pH 2.5 with phosphoric acid. Detection was by UV spectrophotometry at 210 nm. The method has been shown to be selective, precise and rapid and, in a collaborative study, to give excellent correlation with the results obtained by using the biological assay method of the European Pharmacopoeia. The method, which has been applied successfully to the assay of different batches of salmon calcitonin in bulk drug and in formulated products, is recommended for adoption as the pharmacopoeial assay method.     

High-pressure liquid chromatographic assay of benzoyl peroxide in dermatological gels and lotions.

A high-pressure liquid chromatographic method for the assay of benzoyl peroxide in dermatological preparations is described. Degradation products such as benzoic acid and perbenzoic acid do not interfere. The method is simple, precise, accurate, and stability indicating.     

Analysis and stability of the candidate sulfur mustard decontaminant S-330

The chloroamide compound 1,3,4,6-tetrachloro-7,8-diphenyl-2, 5-diiminoglycoluril (S-330) was found to be a strong reactant in dermal formulations for the decontamination of sulfur mustard (HD). In this report, we present analytical methodologies applicable to the characterization, purity determination and quantitation of S-330 in bulk material or formulations. High-performance liquid chromatography-mass spectrometry (LC-MS) coupled with atmospheric pressure chemical ionization (APCI) interface or ultraviolet detector and nuclear magnetic spectroscopy (NMR) were used to identify and characterize S-330 and impurities in the synthetic lots or degradation products in formulations. Bulk synthesis using a chlorination process has yielded a product of 90% purity. The major impurity has been separated and identified structurally as the trichloro analog of S-330. Higher purity S-330 can be made using column chromatography, but this does not appear to be economical for large-scale production. Factors affecting the stability of S-330 in topical formulations include water content, pH, alcohols and UV light. Chloroamide S-330 decomposes at 50-60 degrees C and is not amenable for GC analysis. The HPLC technique is superior to NMR or active chlorine assay in the purity determination for S-330 in bulk material or formulations. In topical formulations containing S-330, 5-10% of water can be tolerated, but alcohols and acidic and basic conditions should be avoided.     

硫乳膏含量测定方法的改进

目的: 改进硫乳膏含量测定的方法。方法: 采用正交试验设计,以硫含量和反应完全程度为考察指标,考察乙醇、液状石蜡和回流时间对结果的影响。结果: 最佳处理方法为加入乙醇3 mL,液状石蜡3 mL,共同回流1 h,改进后的检测方法空白无干扰,平均回收率为99.18%(RSD=0.78%)。结论: 本法简便、快捷,结果准确,可作为硫乳膏的含量测定检测方法。